The generation of new blood vessels, or angiogenesis, plays a key role in the growth of malignant disease and has generated much interest in developing agents that inhibit angiogenesis (see for example Holmgren, L., O'Reilly, M. S. & Folkman, J. (1995) “Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression”, Nature Medicine 1, 149-153; Folkman, J. (1995) “Angiogenesis in cancer, vascular, rheumatoid and other disease”, Nature Medicine 1, 27-31; O'Reilly, M. S., et al., (1994) “Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma”, Cell 79, 315-328; Kerbel, R. S. (1997) “A cancer therapy resistant to resistance”, Nature 390, 335-336; Boehm, T., et al., (1997) “Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance”, Nature 390, 404-7; and Volpert, O. V., et al., (1998) “A human fibrosarcoma inhibits systemic angiogenesis and the growth of experimental metastases via thrombospondin-1”, Proc. Natl. Acad. Sci. (U.S.A.) 95, 6343-6348).
The use of αvβ3 integrin antagonists to inhibit angiogenesis is known in methods to inhibit solid tumor growth by reduction of the blood supply to the solid tumor. See, for example, U.S. Pat. No. 5,753,230 (Brooks & Cheresh) and U.S. Pat. No. 5,766,591 (Brooks & Cheresh) which describe the use of αvβ3 antagonists such as synthetic polypeptides, monoclonal antibodies and mimetics of αvβ3 that bind to the αvβ3 receptor and inhibit angiogenesis.
In addition, antibody-cytokine fusion protein therapies have been described which promote immune response-mediated inhibition of established tumors such as carcinoma metastases. For example, the cytokine interleukin 2 (IL-2) has been fused to a monoclonal antibody heavy chain immunoreactive with, in two separate fusion proteins, the tumor associated antigens epithelial cell adhesion molecule (Ep-CAM, KSA, KS1/4 antigen) or the disialoganglioside GD2 by use of the antibodies KS1/4 and ch14.18, respectively, to form the fusion proteins ch14.18-IL-2 and KS1/4-IL-2, respectively. See, for example, U.S. Pat. No. 5,650,150 (Gillies).
The identification of vasculature-specific inhibitors of angiogenesis that are synergistic with therapies specifically targeting the tumor compartment, will allow for tailoring optimally effective cancer treatment.
Angiogenesis is characterized by invasion, migration and proliferation of endothelial cells, processes that depend on cell interactions with extracellular matrix components. In this context, the endothelial adhesion receptor of integrin αvβ3 was shown to be a key player by providing a vasculature-specific target for anti-angiogenic treatment strategies. (Brooks, P. C., Clark, R. A. & Cheresh, D. A. (1994) “Requirement of vascular integrin alpha v beta 3 for angiogenesis”, Science 264, 569-571; Friedlander, M., et al., (1995) “Definition of two angiogenic pathways by distinct alpha v integrins”, Science 270, 1500-1502). The requirement for vascular integrin αvβ3 in angiogenesis was demonstrated by several in vivo models where the generation of new blood vessels by transplanted human tumors was entirely inhibited either by systemic administration of peptide antagonists of integrin αvβ3 or anti-αvβ3 antibody LM609. (Brooks, P. C., et al., (1994) Science supra; Brooks, P. C., et al., (1994) “Integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels”, Cell 79, 1157-1164). Murine hybridoma LM609 has been deposited with the American Type Culture Collection (ATCC, Manassas, Va., USA) as the International Depository Authority under the Budapest Treaty, and assigned the ATCC Designation HB 9537, on Sep. 15, 1987. Such antagonists block the ligation of integrin αvβ3 which promotes apoptosis of the proliferative angiogenic vascular cells and thereby disrupt the maturation of newly forming blood vessels, an event essential for the proliferation of tumors.
Vascular Endothelial Growth Factor (VEGF) has been identified as a selective angiogenic growth factor that can stimulate endothelial cell mitogenesis. Human tumor biopsies exhibit enhanced expression of VEGF mRNAs by malignant cells and VEGF receptor mRNAs in adjacent endothelial cells. VEGF expression appears to be greatest in regions of tumors adjacent to avascular areas of necrosis. (for review see Thomas et al., (1996) “Vascular Endothelial Growth Factor, a Potent and Selective Angiogenic Agent”, J. Biol. Chem. 271(2): 603-606). Effective anti-tumor therapies may utilize targeting VEGF receptor for inhibition of angiogenesis using monoclonal antibodies. (Witte L. et al., (1998) “Monoclonal antibodies targeting the VEGF receptor-2 (Flk1/KDR) as an anti-angiogenic therapeutic strategy”, Cancer Metastasis Rev. 17(2):155-61.
A major obstacle for effective treatment of disseminated malignancies includes minimal residual disease characterized by micrometastases that lack a well-established vascular supply for delivery of therapeutics. In this regard, a novel immunotherapeutic strategy proved efficient in using tumor compartment-specific monoclonal antibodies to direct cytokines to the tumor microenvironment. This was achieved by recombinant antibody-cytokine fusion proteins, generated to maintain the unique tumor-specific targeting ability of monoclonal antibodies and the immunomodulatory functions of cytokines. The use of an antibody-IL-2 fusion protein to direct IL-2 into the tumor compartment induced activation of effector cells invading the tumor microenvironment and resulted in efficient eradication of established micrometastases in three different syngeneic mouse tumor models. (Becker, J. C., et al. (1996) “T cell-mediated eradication of murine metastatic melanoma induced by targeted interleukin 2 therapy”, J—Exp. Med 183, 2361-2366; Xiang, R., et al., (1997) “Elimination of established murine colon carcinoma metastases by antibody-interleukin 2 fusion protein therapy”, Cancer Res. 57, 4948-4955; Lode, H. N., et al., (1998) “Natural killer cell-mediated eradication of neuroblastoma metastases to bone marrow by targeted interleukin-2 therapy”, Blood 91, 1706-1715). Although quite effective at early stages of tumor metastasis, this tumor compartment-directed approach could only delay growth of metastases at later stages of tumor growth characterized by a fully developed vascular compartment. Here, we addressed the question of whether there is a complementary advantage of specific vascular and tumor compartment-directed treatment strategies being synergistic when used in sequential and simultaneous combinations.
This was tested in three syngeneic murine tumor models of colon carcinoma, melanoma and neuroblastoma, the latter characterized by spontaneous hepatic metastases. All three models exhibit close similarities to the diseases in humans. The melanoma and neuroblastoma models express disialoganglioside GD2, a well-established tumor-associated antigen in such neuroectodermal malignancies (Irie, R. F., Matsuki, T. & Morton, D. L. (1989) “Human monoclonal antibody to ganglioside GM2 for melanoma treatment”, Lancet 1, 786-787; Handgretinger, R., et al., (1995) “A phase I study of human/mouse chimeric antiganglioside GD2 antibody ch14.18 in patients with neuroblastoma”, Eur. J. Cancer 31A, 261-267) and the colon carcinoma model is characterized by the expression of the epithelial cell adhesion molecule (Ep-CAM, KSA, KS1/4 antigen), a target molecule successfully exploited for passive immunotherapy in man (Riethmuller G., et al., (1994) “Randomised trial of monoclonal antibody for adjuvant therapy of resected Duke's C colorectal carcinoma”, Lancet 343, 1177-1183). These antigens specifically delineate the tumor compartment in these models targeted by the antibody-interleukin-2 fusion proteins with human/mouse chimeric anti-GD2 antibody (ch14.18-IL-2)(Gillies, S. D., et al., (1992) “Antibody-targeted interleukin 2 stimulates T-cell killing of autologous tumor cells”, Proc. Natl. Acad. Sci. (U.S.A.) 892, 1428-1432) and humanized anti-Ep-CAM (anti-KSA, anti-KS1/4 antigen) antibody KS1/4-IL-2 (Xiang, R., et al. (1997) supra.; Gillies, S., et al, (1998) “Antibody-IL-12 fusion proteins are effective in SCID mouse models of prostrate and colon carcinoma metastases”, J Immunol. 160, 6195-6203). The vascular compartment of these tumor models, as described in several animal models, is defined by expression of integrin αvβ3 on newly formed blood vessels. (Brooks, P. C., et al., (1994) supra). The data presented here demonstrate a synergistic efficacy of simultaneous and sequential treatments specifically targeting tumor and vascular compartments of primary tumors and distant metastases. A mechanism for this synergism is provided by a decrease in blood vessel formation and an increase in inflammation only in animals treated with the combination therapy. These observations emphasize the beneficial effect of combining anti-angiogenic with tumor-specific anti-tumor immunotherapeutic approaches.